DESCRIPTION (adapted from the Abstract): The intracellular expression of RNA-based inhibitors of HIV-1 functions is potentially a powerful tool by which gene therapy might be used against viral replication. Possible inhibitors include antisense RNAs, catalytic RNAs (ribozymes) designed to cleave viral RNA, or high-affinity RNA ligands (aptamers) designed to bind and inhibit the function of essential viral macromolecules. In preliminary experiments, "decoy" RNAs meant to serve as competitive inhibitors of the essential HIV-1 RNA binding protein Rev have proven to be effective. The potential targets for such RNA inhibitors are not limited to proteins that normally have a high affinity for RNA, however. The advent of in vitro evolution (or SELEX) technology makes possible the selection of a series of high-affinity RNA ligands (aptamers) to multiple epitopes on macromolecular antigens. Simultaneous expression of multiple aptamer inhibitors of an array of viral functions could, in principle, overcome mutational escape by the virus. In the proposed research, the Principal Investigator addresses major problems to be solved using such an approach. These are: (1) identification of additional expression strategies that give efficient synthesis of stable aptamers in diverse subcellular locations in human cells; and (2) matching appropriate expression strategies with existing and newly devised anti-HIV-1 RNAs to allow cells to synthesize their own viral inhibitors in vivo. Expression of anti-HIV-1 aptamers will be tested for the ability to suppress infection in stably transfected cell lines. Effective strategies will be optimized and combinatorial expression of the small RNA inhibitors will be tested for synergistic inhibition of viral infection.